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Instituto de Investigaciones Biomédicas Alberto Sols, Consejo Superior de Investigaciones Cientificas (CSIC) and Universidad Autonoma de Madrid (UAM), 28029 Madrid, Spain
Address all correspondence and requests for reprints to: Dr. Juan Bernal, Instituto de Investigaciones Biomédicas, Arturo Duperier 4, 28029 Madrid, Spain. E-mail: jbernal{at}iib.uam.es
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RC3 expression is physiologically and developmentally regulated. In rats, thyroid hormone (T3) is an important physiological regulator (15, 16). Hypothyroid animals show reversibly decreased concentrations of RC3 messenger RNA and protein in discrete areas of the forebrain. Regulation of RC3 expression by thyroid hormone occurs in developing as well as adult animals (17) and correlates to the known morphogenetic actions of thyroid hormone on dendritic spines of cerebral cortex and hippocampal neurons (18, 19). Regulation by T3 occurs in other species besides the rat. Thus, the goat RC3 homolog has also shown to be thyroid hormone dependent in vivo (20). In addition, in mouse-derived hypothalamic GT1–7 cells, thyroid hormone induces an increased RC3 expression, which occurs at the transcriptional level and is independent of new protein synthesis (21). RC3 regulation by T3 is therefore presumably mediated through a direct interaction of thyroid hormone receptors with regulatory regions in the RC3 gene.
In brain as well as in other tissues, T3 acts in the cell nucleus after binding to specific receptors. These receptors are members of a large family of transcription factors that includes the receptors for other hydrophobic ligands, such as retinoids, steroid hormones, and other hormones or metabolites. They control important developmental and physiological processes by modulating the expression of genes after binding to specific DNA sequences, known as response elements, that are usually located in the upstream promoter region of the target genes (22, 23, 24). Most of these nuclear receptors usually bind to DNA as homodimers or heterodimers with the 9-cis-retinoic acid receptor (RXR) (25). In the case of thyroid hormone, the response elements, or T3REs, usually consist of a direct repeat of the consensus hexamer sequence, AGGTCA, separated by four nucleotides (DR4), although other configurations are also relatively common (26, 27).
We have previously reported the identification, cloning, and determination of the structure of the human RC3 homolog gene, which we have named NRGN (28). The gene spans 12 kb and contains four exons and three introns. Its organization is identical to that of the rat Ng/RC3 gene, and alignment of protein sequences revealed a 97.5% identity. Besides characterization of the gene and chromosomal mapping, few data are available on NRGN expression in humans (29). We have observed that its expression in the monkey brain follows a pattern similar to that previously described for the rat or goat brain (results to be published). This suggests that NRGN could also be under thyroid hormone regulation in primates. To try to answer this question as well as to disclose the mechanism of regulation by T3, we have searched for T3REs in the human NRGN gene. Our previous studies failed to disclose any such regulatory elements in the 5'-flanking region of the rat or human genes (21, 28, 30). In the present work we have used an immunoprecipitation technique that allowed the screening of the whole gene. As a result we report on the localization and characterization of a T3RE in the first intron of the NRGN gene. The sequence consists of a typical DR4, which binds the RXR-T3R heterodimer and is trans-activated in vitro by thyroid hormone when fused to either the homologous promoter or to thymidine kinase promoter. The results provide an explanation for T3 regulation. In addition, they suggest that, as previously shown in the rat and goat, the NRGN gene is also under thyroid hormone regulation, probably contributing to the effects of this hormone on the development and function of the human brain.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-1 cloned in pCDM8 (a gift from Dr. P.
R. Larsen, Brigham and Women’s Hospital, Boston, MA); and retinoid X
receptor expression vector, the human RXR complementary DNA, cloned
in the plasmid pSG-5 (supplied by Dr. H. Stunnenberg, University of
Nijmegen, The Netherlands). The oligonucleotides used in this study
were synthesized as two complementary strands with 5'-extensions
according to the sequence of the NRGN
T3RE, as shown in this paper, and the
T3RE present in the long terminal repeat of the
Moloney murine leukemia virus (Mo) (31). The sequences of these
nucleotides were as follows (the DR4 half-sites are
underlined): NRGN T3RE,
5'-TCGACTTCCAAAATGGGGATTAAATGAGGTAATATC
3'; and Mo T3RE,
5'-TCGACAGGGTCATTTCAGGTCCTTGC-3'. The nonspecific oligonucleotide (NS) used for the gel retardation experiments consisted of a 43-mer oligonucleotide corresponding to a region of the promoter of rat proteolipid protein with the following sequence: 5'-GATCCAAGAGTTTGACTGGCTGATTTCCAGTTTGTG-3'.
The mutated oligonucleotides of the NRGN T3RE were the following (the nucleotide changes are underlined): M1, 5'-TCGACTTCCAAAATGGTTATTAAATGAGGTAATATC 3'; M2, 5'-TCGACTTCCAAAATGGGGATTAAATGATTTAATATC 3'; and M3, 5'-TCGACTTCCAAAATGGGGATTACTTAAGGTAATATC 3'.
The labeled DNA used in the footprinting assay was amplified using the universal primer of the pMosblue vector (Amersham Pharmacia Biotech, Rainham, Essex, UK) (-40: 5'-GTTTTCCCAGTCACGAC-3') and the internal primer of the NRGN fragment (1588: 5'-TGACT-TTTCCTGCATTCAC-3').
For trans-activation experiments, a single copy of each of the following oligonucleotides, NRGN T3RE, Mo T3RE, M1, M2, and M3, was subcloned into the SalI site of pBLCAT-2 vector, which contains the CAT gene under the control of the thymidine kinase promoter. In addition, to evaluate the ability of the NRGN T3RE to regulate the activity of the homologous promoter, the T3RE was inserted immediately upstream of the NRGN promoter [nucleotides -513 to +76 (28)] in the promoterless pBLCAT-3 vector.
Transfection and CAT assays
COS-7 cells were grown and maintained in DMEM supplemented with
10% FBS. The cells were plated to a density of 250,000 cells/6-cm
plate the day before transfection. The cells were transfected by the
calcium phosphate protocol (32), using 5 µg of the appropriate CAT
construct, 0.3 µg of the expression vector containing the nuclear
receptor, and 4 µg of the plasmid pCH110 (Amersham Pharmacia Biotech). This plasmid contains the structural gene for
ß-galactosidase under control of the simian virus 40 early promoter
and was used as an internal control of transfection efficiency. Sixteen
to 18 h after DNA addition the cells were washed with PBS and
replenished with medium containing serum depleted of thyroid hormones
by treatment with Dowex resin. Where appropriate,
T3 (Sigma Chemical Co., St. Louis,
MO) was added to a concentration of 150 nM, and the cells
were incubated for 24 h before harvesting for determination of
ß-galactosidase and CAT activities (33).
Isolation of T3R-bound genomic DNA
fragments
To isolate DNA fragments able to bind T3R
we followed an immunoprecipitation method described previously (34).
Plasmid clones spanning the whole NRGN gene (28) were
cleaved with different restriction enzymes, and all of the resulting
fragments were end labeled with Klenow DNA polymerase and
[32P]deoxy-CTP. T3Rß1 and RXR
were obtained by in vitro translation using the TNT kit (TNT
Sp6 Coupled Reticulocyte Lysate System, Promega Corp.,
Madison, WI). The reaction mixture contained 105 cpm
(4 x 107 cpm/µg) labeled DNA and 10–20 fmol of the
in vitro translated T3Rß and RXR
in 40 µl binding buffer [25 mM HEPES (pH 7.9), 100
mM KCl, 1 mM dithiothreitol (DTT), 10%
glycerol, and 0,05 mg/ml poly(dI-dC)]. After incubation for 30 min at
room temperature, 500 ng anti-T3R antibody were
added, and incubation was continued for 30 min at room temperature. To
recover the immunocomplexes, 20 µl 50% protein A-Sepharose,
previously equilibrated in binding buffer, were added and mixed during
20 min at room temperature on a rotating wheel.
Protein A-Sepharose was washed three times with 500 µl washing buffer [25 mM HEPES (pH 7.9), 100 mM KCl, 1 mM DTT, and 10% glycerol], and the precipitated DNA fragments were eluted with 100 µl 1 M NaCl, extracted with phenol-chloroform, and precipitated with 100 µl isopropanol and 5 µg transfer RNA. The pellet was resuspended in 15 µl TE [10 mM Tris-HCl (pH 8) and 1 mM EDTA (pH 8)] and run on a 1% agarose gel, which was then fixed in 10% trichloroacetic acid and dried. The gel was exposed for 12 h at -70 C.
Footprinting assays
Deoxyribonuclease I (DNase I) footprinting assays were performed
with 3 ng (4 x 105 cpm) 32P-labeled,
260-bp BamHI-EcoRV immunoprecipitated fragment,
subcloned in pMosblue. The labeled probe was obtained by PCR
amplification using the universal primer of the vector, labeled at one
end with T4 polynucleotide kinase, plus an unlabeled internal primer of
the fragment. The PCR conditions were: 5 min at 94 C; 35 cycles of 30
sec at 94 C, 30 sec at 55 C, and 1 min at 72 C; and 7 min
at 72 C. Nuclear extracts from HeLa cells infected with vaccinia virus
expression vectors for the T3R and RXR (a
gift from Dr. H. Stunnenberg) were incubated in 40 µl binding buffer
[25 mM HEPES (pH 7.9), 100 mM KCl, 1
mM DTT, 10% glycerol, and 0.05 mg/ml poly(dI-dC)]. After
5-min incubation at room temperature, the radioactive probe was added
and incubated for a further 30 min. Increasing amounts of DNase I were
then added, and incubation was continued for 1 additional min. The
reaction was terminated on ice by adding stop buffer (25 mM
EDTA and 1% SDS). Samples were extracted with phenol/chloroform
and ethanol precipitated. Pellets were resuspended in 6 µl water
and 5 µl formamide loading buffer (80% formamide, 20 mM
EDTA, 0.025% bromophenol blue, and 0.025% xylene-cyanol) and boiled
for 3 min before loading on a 8% polyacrylamide-7 M urea
sequencing gel. Electrophoresis was carried out at room temperature in
1 x Tris-borate buffer (89 mM Tris base, 88
mM boric acid, and 2 mM EDTA). As a control, 6
µg BSA, instead of nuclear receptors, were used in parallel
reactions.
Electrophoretic mobility shift assays (EMSA)
To study the interaction of T3R and RXR
proteins with DNA, we used in vitro translated nuclear
receptors. Complementary oligonucleotides were annealed, labeled with
T4 polynucleotide kinase and [
-32P]ATP, and purified
with QIAquick Nucleotide Removal Kit (Qiagen, Chatsworth,
CA). The translated receptors were incubated in 13 µl binding buffer
[15% glycerol, 5 mM MgCl2, 50 mM
KCl, 20 mM HEPES (pH 7.9), 5 mM DTT, and 0.3
mg/ml poly(dI-dC)] at room temperature. After 15 min, labeled probes
(4 x 104 cpm) were added to the binding mixture, and
incubation was continued for 15 min. Competition gel-shift studies were
carried out by adding the indicated amounts of the competitor
oligonucleotide at the same time as the probe. The protein-DNA
complexes formed during binding reactions were separated on 6%
polyacrylamide gels in 0.25 x Tris-borate buffer at 250 V. The
gels were dried and exposed to films.
Supershift analysis was carried out by the addition of 1 µl of an anti-T3R antibody to the gel shift reaction after the 30-min incubation. The reaction mixture was incubated on ice for an additional 30 min and then loaded on the gel.
For off-rate experiments, a 200-fold molar excess of unlabeled oligonucleotides was added after the binding reaction. Samples were withdrawn at intervals between 0–45 min and directly loaded on a running gel. These experiments were performed in the presence of either 1 mM EDTA or 1 mM MgCl2 in the binding buffer (35).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We followed an immunoprecipitation technique to isolate genomic
fragments able to bind the T3 receptor protein
(34). NRGN genomic clones were cleaved by a set of
restriction enzymes, followed by end labeling of the resulting DNA
fragments. The labeled fragments were then used in
coimmunoprecipitation assays in an attempt to identify
T3REs on the basis of their expected interaction
with the receptor. For immunoprecipitations, we used a mixture of
T3Rß and RXR proteins, previously obtained
in vitro using a coupled transcription and translation
rabbit reticulocyte lysate system. Control samples contained
unprogrammed reticulocyte lysate. The mixture of labeled DNA fragments
was incubated with either control or
T3R/RXR-programmed lysate proteins, and the
resulting complexes were immunoprecipitated with a specific
anti-T3R antibody. Parallel incubations were
performed in the presence of an excess of a DR4
T3RE, like the one present in the Moloney murine
leukemia virus promoter, to determine the absence of nonspecific
binding to DNA.
Figure 1A
shows the structure of the
human gene based on the report by Martinez de Arrieta et al.
(28). The gene contains four exons and three introns. Exon 1 encodes
the 5'-untranslated region and the first five amino acids of the
protein sequence; the rest of the NRGN protein is encoded in exon 2.
Exons 1 and 2 are interrupted by an intron of about 5 kb long. In this
work, we used several DNA fragments of sizes up to 1.5 kb, encompassing
the human gene from about 3 kb upstream from the initiation of
transcription to 0.5 kb downstream from the end of the fourth exon. All
of these genomic fragments were negative in the immunoprecipitation
assays, except for a 1.5-kb fragment of the first intron of the gene
from approximately nucleotide 3000–4500 (Fig. 1A) from the origin of
transcription. This fragment was cloned in pMosblue and subjected to
further restriction digestion and immunoprecipitation analysis to more
precisely delineate the T3R-interacting region.
Figure 1A shows a restriction enzyme map, and Figure 1B shows the
result of immunoprecipitation. Digestion with XbaI plus
EcoRI produced four fragments: two of 0.1 kb, one of 1.4 kb,
and one of 2.8 kb, the latter containing the vector. The labeled
fragments were resolved in agar electrophoresis as three bands (Fig. 1B, lane D). After incubation with T3R/RXR plus
an anti-T3R antibody (lane R), only the 1.4-kb
band was precipitated. The specificity of this effect was confirmed by
performing a similar coimmunoprecipitation experiment using as
unlabeled competitor an excess of the T3RE
present in the Moloney murine leukemia virus (lane Mo). In addition,
the band was not observed if an unprogrammed reticulocyte lysate (lane
RL) was used. Digestion with BamHI produced three bands,
consisting of the vector plus two bands of 0.9 and 0.6 kb,
respectively. After incubation with receptors and the
anti-T3R antibody, only the 0.6-kb band was
significantly immunoprecipitated. Further digestion and
immunoprecipitation of smaller fragments (not shown) narrowed the size
range of the DNA able to bind T3R/RXR to a 260-bp
BamHI-EcoRV fragment derived from the 5'-portion
of the original fragment.
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and RXR,
plus increasing amounts of DNase I (Fig. 2A) resulted in a major protected
sequence of 20 nucleotides. Control reactions performed in the presence
of BSA showed no protected sequence. Analysis of the protected sequence
shown in Fig. 2A reveals an imperfect direct repeat of the consensus
half-site AGGTCA separated by four nucleotides (GGATTAaatgAGGTAA).
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). For this
purpose, either a 140-bp fragment from the NRGN first intron
containing the T3RE or a single copy of a double
stranded oligonucleotide (NRGN T3RE, as shown in
Materials and Methods) was cloned upstream from the
thymidine kinase promoter in the pBLCAT-2 vector (140-tk-CAT or
NRGN-tk-CAT, respectively). COS-7 cells were cotransfected with either
of these two constructs together with expression vectors encoding
T3R and RXR. For comparison, cells were similarly
cotransfected with the CAT expression vector containing a single copy
of the strong T3RE present in the Moloney murine
leukemia virus long terminal repeat, a prototypical DR4 (Mo-tk-CAT).
CAT activity was measured after incubating the cells in the absence or
presence of T3. The cells transfected with either
NRGN construct responded to the addition of T3
with an increased CAT activity (Fig. 2B, upper panel). In
addition, we checked whether the T3RE was able to
confer regulation in the context of the NRGN promoter. For this
purpose, either the Moloney DR4 or the NRGN T3RE
was inserted upstream of the NRGN promoter in the promoterless pBLCAT-3
vector (Mo-0.6-CAT and NRGN-0.6-CAT, respectively). In both constructs
the addition of T3 increased CAT activity,
whereas it did not when the 0.6-CAT construct without a
T3RE was used as the control (Fig. 2B, lower panel).
Interaction of the thyroid hormone receptor with the NRGN
T3RE
A set of experiments was designed to characterize the interaction
of T3R in vitro with the NRGN
T3RE by EMSA, using in vitro
translated T3Rß and RXR, and labeled
oligonucleotides. For comparison, we also used the Moloney
T3RE (Fig. 3A).
When labeled NRGN T3RE oligonucleotide was
incubated with human T3Rß and RXR (lane 2),
a retarded band was observed showing the same mobility shift than the
RXR-T3R heterodimer obtained with the Mo
T3RE (lane 1). Neither T3R
(lanes 6 and 8) nor RXR (lane 9) alone bound the
T3RE. The specificity of receptor binding was
determined by the addition of an excess of unlabeled competitors. The
addition of Mo and NRGN oligonucleotides (lanes 3 and 5) completely
avoided formation of the retarded band. The complex was not competed,
however, when incubated in the presence of a random DNA sequence (lane
4). Further proof of the binding of T3R to the
NRGN oligonucleotide was obtained in supershift experiments, as shown
in Fig. 3B. In these experiments, the same shifted band was produced
with T3R/RXR plus either Mo (lane 10) or NRGN
(lane 12) oligonucleotides. The NRGN band was supershifted by addition
of an anti-T3R antibody to the incubation mixture
(lane 11), whereas the antibody alone was without effect (lane 13).
These experiments suggested that the NRGN T3RE
bound the RXR-T3R heterodimer and had no affinity
for either T3R monomer or homodimer.
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). These experiments
were performed in the presence or absence of Mg2+ (35). In
the absence of Mg2+, the complex formed on the Mo
T3RE was very stable, with almost no dissociation
after 45 min, a period during which about 20% of the NRGN-receptor
complex was dissociated. In the presence of Mg2+, the
half-life of the Mo complex was about 5 min, and that of the NRGN
complex was 2 min.
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). The oligonucleotides were labeled
and incubated with receptors in the presence or absence of the
homologous unlabeled oligonucleotide in a molar excess to determine the
specificity of the retarded complex (Fig. 5B). The substitution of two
thymidines for two guanines in the first half-site (M1) reduced the
ability to bind the receptors. The same mutation in the second
half-site (M2) completely suppressed the formation of the retarded
band. Exchange of the wild-type spacer sequence for another functional
sequence, CTTA (M3) (38), produced a similar retarded band as with the
NRGN oligonucleotide. In Fig. 5C, the labeled NRGN oligonucleotide was
incubated with increasing amounts of competing oligonucleotides, such
as the wild-type sequence; the mutants M1, M2, and M3; and a
nonspecific sequence (NS). Consistent with the results shown in Fig. 5B, when the wild-type sequence and that with the mutated spacer (M3)
were used, a small excess of cold oligonucleotide strongly decreased
the labeled DNA-protein complex, whereas a nonspecific sequence had no
competitor activity. Modification of the first half-site (M1) decreased
the competing effect, but did not obliterate it completely. In
contrast, when the second half-site was modified (M2), the competing
effect of the oligonucleotide was completely lost. These results
suggest that the integrity of the second half-site is essential for
binding the RXR-T3R heterodimer.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The presence of a T3RE in the first intron of the gene, as shown here, strongly suggests that NRGN is a target of thyroid hormone in the human brain. The T3RE was localized using a previously described technique that allowed the isolation of T3 target genes from genomic libraries (34, 44). Further characterization of the T3RE sequence was carried out by footprinting, EMSA, and trans-activation analysis. The footprinted sequence from NRGN contains an imperfect DR4 sequence (ggGGATTAaatgAGGTAA), with the second half-site almost identical to the consensus sequence. It binds the RXR-T3R heterodimer, but not T3R monomers or dimers, as with other DR4 sequences that contain guanosine in the spacer sequence between the half-sites and lack TC or TA motifs upstream of the first half-site (25). The half-life of the complex is similar to that of the strong T3RE present in the Moloney virus terminal repeat, and the protein complex shifted in the EMSA experiments was supershifted with an anti-T3R antibody. Mutations of the two half-sites revealed the requirement for the integrity of the second half-site to bind the complex, in agreement with the known polarity of the RXR-T3R heterodimer binding to DNA. Current models suggest that T3R binds first to the distal half-site, and subsequent binding of RXR increases binding affinity (45, 46).
Interestingly, NRGN T3RE is located in the first intron of the gene, and preliminary data from our laboratory suggest that a T3R-binding site is located in a similar position in the first intron of the rat RC3 gene, in a conserved region of the intron showing a high degree of similarity to the human gene. Although most of the reported T3REs are located in the upstream promoter region of the target genes, T3REs in intronic locations have also been described, such as in the rat GH gene, NCAM, and Pcp2 (39, 47, 48). Among the thyroid hormone-dependent genes in brain only a few of them have been shown to contain T3REs. These include myelin basic protein (49), NCAM (47), tubulin (47), Pcp2 (48), and PGD2 synthase (50). NRGN is the second human brain gene, after PGD2 synthase (51), in which a T3RE has been identified.
Previous studies in the rat have shown that the control exerted by thyroid hormone on RC3 expression is region specific (16). Some neuronal populations were sensitive, whereas others insensitive to the effects produced by either thyroid hormone deprivation or supplementation. The sensitive regions were layer 6 of the cerebral cortex, layers 2–3 of retrosplenial cortex, the lateral caudate, and the granular layer of the hippocampus. The insensitive regions included, among others, the upper layers of cerebral cortex, the pyramidal layers of the hippocampus, and the amygdala. The fact that RC3 expression is modulated by thyroid hormone in specific regions suggested that the T3 control was indirect, so that RC3 could be a distal response subsequent to activation of an earlier responsive gene. However, as pointed out above, T3 induces RC3 expression in cultured cells at the transcriptional level and does not require protein synthesis (21). The finding of a T3RE in NRGN provides further support for a direct interaction of thyroid hormone receptors with regulatory sequences in this gene. Therefore, the reasons for differential T3 sensitivity remain to be determined. We have previously discarded the idea that this phenomenon is due to differential expression of thyroid hormone receptor isoforms (36). Other possibilities to be explored are differential expression of T3R coregulators or an effect due to chromatin conformation.
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Acknowledgments |
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Footnotes |
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2 C.M.A. and B.M. contributed equally to this work.
3 Predoctoral fellow supported by the Basque Government.
4 Postdoctoral fellow supported by the Community of Madrid.
In this article we refer to RC3 as the rat RC3/neurogranin gene, and to NRGN as the human gene.
Received May 8, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2 is a weak
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3000–4500) bound in
the coimmunoprecipitation assays is shown in expanded scale, and the
restriction sites are also indicated (B, BamHI; E,
EcoRI; RV, EcoRV; X,
XbaI); the smaller immunoprecipitated fragment is
represented by the shaded area. B, The plasmid
containing a 1.5-kb genomic fragment of NRGN was
digested with XbaI+EcoRI and
BamHI, and the resulting fragments were end labeled with
Klenow enzyme. Lane D shows the labeled fragments before the
immunoprecipitation assays. The fragments were incubated with
T3R/RXR proteins (lane R) and were competed with a 200-fold
increase in unlabeled T3RE present in the Moloney murine
leukemia virus (lane Mo). As a control, an incubation with an
unprogrammed reticulocyte lysate (lane RL) was included. Complexes were
then precipitated with an anti-T3R antibody bound to
protein A-Sepharose.
,
Mg2+ binding buffer; •, EDTA binding buffer.